A plant-based oral vaccine to protect against systemic intoxication by Shiga toxin type 2

Abstract

Hemolytic uremic syndrome, the leading cause of kidney failure in children, often follows infection with enterohemorrhagic Escherichia coli and is mediated by the Shiga type toxins, particularly type 2 (Stx2), produced by such strains. The challenge in protecting against this life-threatening syndrome is to stimulate an immune response at the site of infection while also protecting against Shiga intoxication at distal sites such as the kidney. As one approach to meeting this challenge, we sought to develop and characterize a prototypic orally delivered, plant-based vaccine against Stx2, an AB5 toxin. First, we genetically inactivated the Stx2 active A subunit gene and then optimized both subunit genes for expression in plants. The toxoid genes were then transformed into the Nicotiana tabacum (tobacco) cell line NT-1 by Agrobacterium tumefaciens-mediated transformation. Toxoid expression was detected in NT-1 cell extracts, and the assembly of the holotoxoid was confirmed. Finally, mice were immunized by feeding with the toxoid-expressing NT-1 cells or by parenteral immunization followed by oral vaccination (prime-boost strategy). The immunized mice produced Stx2-specific mucosal IgA and Stx2-neutralizing serum IgG. The protective efficacy of these responses was assessed by challenging the immunized mice with E. coli O91:H21 strain B2F1, an isolate that produces an activatable variant of Stx2 (Stx2d) and is lethal to mice. The oral immunization fully protected mice from the challenge. Results of this study demonstrated that a plant-based oral vaccine can confer protection against lethal systemic intoxication.

Fig. 1.

Diagram of the plant transformation vectors pSW3 and pSW2. Shown are the genetic elements in pGPTV-Kan between the left border (LB) and right border (RB) sequences that define the transferred DNA. I: npt II, the neomycin phosphotransferase gene for kanamycin resistance, flanked by the nopaline synthase promoter (NOS) and the nopaline synthase polyadenylation signal (Ag7). II: the toxoid stxA_2PO, flanked by the cauliflower mosaic virus (CaMV) 35S RNA promoter, followed by the 5′ untranslated region (UTR) from tobacco mosaic virus (TMVΩ) and the soybean vegetative storage protein (VSP) 3′ polyadenylation sequence. III: an intergenic terminator sequence derived from the potato proteinase inhibitor II gene, pin2. IV: the plant-optimized stxB_2PO (pSW3) or native stxB₂ (pSW2) eukaryotic expression cassette with the tobacco etch virus (TEV) 5′ UTR. Restriction sites used for ligations are indicated below the diagram.

Fig. 2.

Western blot analysis of the Stx2 holotoxoid expressed by transformed NT-1 clones. The positive control consisted of 10 ng of the wild-type Stx2 purified from E. coli DH5α that harbored pMJ100 (39) (lane 1). Sonic lysates of 50 mg of fresh plant cells were used to load lanes 2–4. Lane 2, NT-1Kan^R clone served as the negative control; lane 3, NT#2 clone; and lane 4, NT#3 clone. Stx2A and Stx2B subunit positions are indicated by arrows.

Fig. 3.

Assessment of plant-derived Stx2 holotoxoid assembly by the Gb₃-binding assay. Serially diluted sonic lysates of E. coli DH5α (pMJ100) that expressed wild-type Stx2, NT#3, or NT-1Kan^R were added to Gb₃-coated wells. Wells without Gb₃ were also treated with E. coli DH5α (pMJ100) to serve as controls for nonspecific binding by Stx2. The experiment was done in triplicate, and each datum point represents the mean value of replicates ± 1 SD.

Fig. 4.

Stx2-specific fecal IgA as measured by ELISA. Each circle represents the IgA titer of an individual mouse; the bar represents the geometric mean of the titers obtained from the group. The dashed line indicates the limit of detection, and the error bracket represents the 95% confidence interval. Unpaired t tests were performed between group pairs (see Table 1): A–B (P < 0.05), C–D (P > 0.05), C–E (P > 0.05), and D–E (P > 0.05).

Fig. 5.

Neutralization of Stx2 Vero cell cytotoxicity with mouse antisera. Each circle represents the neutralization titer of the serum from an individual mouse. The dashed line represents the limit of detection. The bar represents the geometric mean of neutralization titers obtained from each group, and the error bracket represents the 95% confidence interval. Unpaired t tests were performed between group pairs (see Table 1): A–B (P < 0.05), C–D (P < 0.05), C-E (P < 0.05), and D–E (P < 0.05).

Fig. 6.

Survival of streptomycin-treated mice after systemic toxin challenge by oral infection with B2F1. Letters in the legend box indicate immunization groups (Table 1). Statistical analysis was done on day 12 after oral infection to compare between groups A–B, C–D, C–E, and D–E by using Fisher’s exact test (P < 0.05).